BK channel malfunction has been implicated in several pathologies such as hypertension, epilepsy, asthma, hearing loss, and tumor growth regulation. The BK channel pore-forming -subunit is almost ubiquitously expressed and the homotetramer of -subunits is the BK channel minimal functional unit. BK channel currents exhibit substantial phenotypic diversity among different tissues challenging the potential use of BK channels as therapeutic targets. Auxiliary subunits, which are tissue-specific expressed proteins, play a major role in defining the functional properties of BK channels. 4 types of -subunits (1-4) and recently 2 types of ?-subunits (?1-?2) have been described as BK auxiliary subunits, each one defining physiologically critical properties of the complex including apparent Ca2+-sensitivity, ability to inactivate, activation/deactivation kinetics and pharmacology. The fac that each type of auxiliary subunit produces different effects raises the question of the functiona consequences of heteromeric assembly of auxiliary subunits in individual channels, but this possibility has never been explored in BK channels. Here, I hypothesize that distinct types of auxiliary subunits can coassemble in a single BK channel and potentially contribute to the BK functional diversity observed in native cells. My preliminary experiments suggest that heteromeric channels containing 2+3a and 2+?1 can occur. But, what are the possible stoichiometries of their coassembly? Are there unique, maybe unexpected functional consequences from such channels? Are they present in native tissues? These issues will be addressed combining functional (single channel recordings) and an optical method (single molecule TIRF). The analysis of single channel recordings will take advantage of unique behavior conferred by each of the auxiliary subunits on a single BK channel. The combinations 2+3a and 2+?1 will be studied in heterologous systems (Xenopus oocytes) and in isolated cells from Vomeronasal Organ of WT and 2-KO mice. Whatever the outcome of these experiments, the results are expected to provide new insight into the diversity of BK channels that can arise from heteromeric auxiliary subunit assembly. PUBLIC HEALTH RELEVANCE: BK channel malfunction has been implicated in pathologies such as hypertension and epilepsy. Understanding BK channel phenotypic diversity is critical for assessing BK channels as potential therapeutic targets. This project research will identify new mechanisms of BK functional diversity arising from auxiliary subunits.